Ambient pressure matrix-assisted laser desorption ionization (MALDI) apparatus and method of analysis

ABSTRACT

A mass spectrometer having a matrix-assisted laser desorption ionization (MALDI) source which operates at ambient pressure is disclosed. The apparatus and method are disclosed to analyze at least one sample which contains at least one analyte using matrix-assisted laser desorption ionization (MALDI), which apparatus comprises:  
     The present invention relates to an apparatus and a method for ionizing at least one analyte in a sample for delivery to a mass analysis device, comprising:  
     (a) an ionization enclosure including a passageway configured for delivery of ions to the mass analysis device;  
     (b) means to maintain said ionization enclosure at an ambient pressure of greater than 100 mTorr;  
     (c) a holder configured for maintaining a matrix containing said sample in the ionization enclosure at said ambient pressure;  
     (d) a source of laser energy including means associated with the ionization enclosure for directing the laser energy onto said matrix maintained by the holder at the ambient pressure to desorb and ionize at least a portion of the analyte in the sample, and  
     (e) means for directing at least a portion of the at least one ionized analyte into the passageway. The ambient pressure (AP-MALDI) source is compatible with various mass analyzers, particularly with mass spectrometers and solves many problems associated with conventional MALDI sources operating under vacuum. Atmospheric pressure MALDI is described. The analysis of organic molecules or fragments thereof, particularly biomolecules, e.g., biopolymers and organisms, is described.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. provisionalpatent application Ser. No. 60/089,088, filed Jun. 12, 1998 which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to the field of mass spectrometry, and moreparticularly to a matrix-assisted laser desorption ionization (MALDI)source for mass spectrometry at about atmospheric pressure. Thisinvention is useful to obtain structural data of compounds especiallylarge complex species.

[0004] 2. Description of Related Art

[0005] A mass spectrometer generally contains the following components:

[0006] (1) an optional device to introduce the sample to be analyzed(hereinafter referred to as the “analyte”), such as a liquid or gaschromatograph, direct insertion probe, syringe pump, autosampler orother interfacing device;

[0007] (2) an ionization source, which produces ions from the analyte;

[0008] (3) at least one analyzer or filter which separates the ionsaccording to their mass-to-charge ratio (m/z);

[0009] (4) a detector which measures the abundance of the ions; and

[0010] (5) a data processing system that produces a mass spectrum of theanalyte.

[0011] There are a number of different ionization sources which arecommonly utilized depending upon the type of analyte, including electronimpact, chemical ionization, secondary ion mass spectrometry(hereinafter referred to as “SIMS”), fast ion or atom bombardmentionization (hereinafter referred to as “FAB”), field desorption, plasmadesorption, laser desorption (hereinafter referred to as “LD”), andmatrix-assisted laser desorption ionization (hereinafter referred to as“MALDI”), particle beam, thermospray, electrospray (hereinafter referredto as “ESI”), atmospheric pressure chemical ionization (hereinafterreferred to as “APCI”), and inductively coupled plasma ionization.

[0012] FAB, ESI and MALDI are particularly useful for the mass analysisand characterization of macromolecules, including polymer molecules,bio-organic molecules (such as peptides, proteins, oligonucleotides,oligosaccharides, DNA, RNA) and small organisms (such as bacteria).MALDI is generally preferred because of its superior sensitivity andgreater tolerance of different contaminants such as salts, buffers,detergents and because it does not require a preliminary chromatographicseparation.

[0013] In the MALDI method, the analyte is mixed in a solvent with smallorganic molecules having a strong absorption at the laser wavelength(hereinafter referred to as the “matrix”). The solution containing thedissolved analyte and matrix is applied to a metal probe tip or samplestage. As the solvent evaporates, the analyte and matrix co-precipitateout of solution to form a solid solution of the analyte in the matrix onthe surface of the probe tip or sample stage. The co-precipitate is thenirradiated with a short laser pulse inducing the accumulation of a largeamount of energy in the co-precipitate through electronic excitation ormolecular vibrations of the matrix molecules. The matrix dissipates theenergy by desorption, carrying along the analyte into the gaseous phase.During this desorption process, ions are formed by charge transferbetween the photoexcited matrix and the analyte.

[0014] The most common type of mass analyzer used with MALDI is thetime-of-flight (hereinafter referred to as “TOF”) analyzer. However,other mass analyzers, such as ion trap, ion cyclotron resonance massspectrometers and quadrupole time-of-flight (QTOF) may be used. Thesemass analyzers must operate under high vacuum, generally less than1×10⁻⁵ torr. Accordingly, conventional MALDI sources have been operatedunder high vacuum. This requirement introduces many disadvantagesincluding inter alia:

[0015] (1) changing the sample holder requires breaking the vacuum whichseverely limits sample throughput and generally requires userintervention.

[0016] (2) the amount of laser energy used must be kept to a minimum toprevent a broadening of the energy spread of the ions which reducesresolution and capture efficiency;

[0017] (3) the positional accuracy and flatness of the sample stage iscritical to the mass assignment accuracy and resolution;

[0018] (4) it is difficult to test analytes directly on surfaces whichare not compatible with high vacuum conditions, including such surfacesas electrophoresis gels and polymer membranes which often shrink underhigh vacuum conditions; and

[0019] (5) tandem mass spectrometry analysis by TOF is relativelydifficult and expensive.

[0020] Thus, it would be advantageous to develop a MALDI which operatesat about atmospheric pressure yet is still compatible with various massanalyzers to solve the above-described problems. However, no one hasheretofore constructed a MALDI source which operates at ambientpressure.

[0021] There have been some efforts by others to develop other types ofionization sources which operate at atmospheric pressure.

[0022] (a) ESI is a method wherein a solution of the analyte isintroduced as a spray into the ion source of the mass spectrometer atatmospheric pressure. The liquid sample emerges from a capillary that ismaintained at a few kilovolts relative to its surroundings, whereby theresultant field at the capillary tip charges the surface of the liquiddispersing it by Coulomb forces into a spray of charged droplets. WhileESI is a powerful ionization method for macromolecules and smallmolecules, it is a dynamic method wherein analyte ions are formed in aflowing electrospray. By contrast, MALDI is a pulsed technique whereinionization of the analyte occurs via a transfer of charge (often aproton) between the absorbing matrix which is irradiated by a pulsedlaser of the proper wavelength. Although the MALDI method is inherentlymore qualitative, its strengths lie in its ability to analyze compoundsdirectly, often in complex biological matrices without extensive samplepreparation and/or prior separation. Moreover, MALDI provides ions oflow charge states, mostly singly and doubly charged quasimolecular ions,whereas electrospray ionization often produces multiple charge states(charge envelope), particularly for large biomolecules such as proteins.

[0023] (b) U.S. Pat. No. 4,527,059 discloses a mass spectrometer havinga sample holder mounted on the outside of the vacuum chamber of a massanalyzer. The sample holder exposes the sample to atmospheric pressureor an inert gas environment and is constructed with a polymer carrierfilm on which the analyte is deposited and which forms part of a wall ofthe vacuum chamber of the mass spectrometer. The laser is directed ontothe analyte causing the analyte to evaporate and simultaneously forminga hole in the carrier film through which the evaporated analyte istransferred into the vacuum chamber. The mass spectrometer uses anionization source which works on a surface-specific basis, such as SIMS,FAB, and a laser-activated micromass analyzer. This is a laserevaporation/ionization device that is not matrix-assisted.

[0024] (c) U.S. Pat. No. 4,740,692 discloses an apparatus using twolasers to produce ions. A first laser is used to vaporize a sample underatmospheric pressure. The second laser is used to ionize the vaporizedsample after the vaporized sample enters the vacuum system. While someof the vaporized sample may ionize when the first laser is used underatmospheric pressure, the ions quickly neutralize from interactions withthe background gas. This is a laser desorption/ionization device that isnot matrix-assisted.

[0025] (d) U.S. Pat. No. 5,045,694 discloses a method and instrument forthe laser desorption of ions in mass spectrometry. The method teachesthe use of matrix compounds which strongly absorb photons from a UVlaser beam operating at wavelengths between 200-600 nm, preferably330-550 nm. Large organic molecules with masses greater than 10,000Dalton to 200,000 Dalton or higher are analyzed with improved resolutionby deflecting low mass (<10,000 Dalton) ions. Both positive and negativeions can be analyzed with reduced fragmentation. The device consists ofa TOF mass spectrometer having a MALDI source with a sample probe thatis inserted into the vacuum chamber of the mass spectrometer. Analyteionization occurs by the MALDI process at the sample probe's tip withinthe vacuum chamber of the mass spectrometer.

[0026] (e) U.S. Pat. No. 5,118,937 discloses a process and device forthe laser desorption of analyte molecular ions, especially biomolecules.Specific matrices and lasers are employed. The device consists of a TOFmass spectrometer having a MALDI source with a specimen support locatedwithin the vacuum chamber of the mass spectrometer or intrinsic to thevacuum chamber wall of the mass spectrometer. Analyte ionization occurswithin the vacuum chamber of the mass spectrometer.

[0027] (f) U.S. Pat. No. 5,663,561 discloses a device and method for theionization of analyte molecules at atmospheric pressure by chemicalionization which includes:

[0028] (l) codepositing the analyte molecules together with adecomposable matrix material (cellulose trinitrate or tr-initrotolueneform a preferred class) on a solid support;

[0029] (2) decomposing the matrix with a laser and thereby blasting theanalyte molecules into the surrounding gas;

[0030] (3) ionizing the analyte molecules within the gas stream by APCIusing reactant ions formed in a corona discharge.

[0031] Unlike MALDI, this method requires that the desorption of theanalyte be carried out as a separate step from the ionization of theanalyte.

[0032] Some other U.S. patents of specific interest include but are notlimited to: Inventor U.S. Pat. No. Issue Date Gray 3,944,826 Mar. 16,1976 Renner et al. 4,209,697 Jun. 24, 1980 Carr et al. 4,239,967 Dec.16, 1980 Brannee et al. 4,259,572 Mar. 31, 1980 Stuke 4,686,366 Aug. 11,1987 Lee et al. 5,070,240 Dec. 3, 1991 Kotamori et al. 5,164,592 Nov.17, 1992 Cottrell et al. 5,260,571 Nov. 9, 1993 Buttrill, Jr. 5,300,774Apr. 5, 1994 Levis et al. 5,580,733 Dec. 3, 1996 Vestal et al. 5,625,184April 29, 1997 Sakain et al. 5,633,496 May 27, 1997

[0033] Other references of interest include:

[0034] M. Karas, et al. International Journal of Mass Spectrometry andIon Processes, 78, (1987) 53-68. “Matrix-Assisted Ultraviolet LaserDesorption of Non-volatile Compounds”.

[0035] K. Tanaka, et al. Rapid Communications in Mass Spectrometry, 2,(1988) 151.

[0036] F. Hillenkamp, Analytical Chemistry, 20, (1988), 2299-3000(Correspondence). “Laser Desorption Ionization of Proteins withMolecular Masses Exceeding 10000 Daltons”.

[0037] M. Karas, et al. International Journal of Mass Spectrometry andIon Processes, 92, (1989) 231-242. “UV Laser MatrixDesorption/Ionization Mass Spectrometry of Proteins in the 100000 DaltonRange”.

[0038] R. Beavis, et al. “Cinnamic Acid Derivatives as Matrices forUltraviolet Laser Desorption Mass Spectrometry of Proteins”. RapidCommunications in Mass Spectrometry, 3, (1989) 432-435.

[0039] M. Karas, et al. Analytica Chimica Acta, 241, (1990) 175-185.“Principles and applications of matrix-assisted UV-laserdesorption/ionization mass spectrometry”.

[0040] A. Overberg, et al. Rapid Communications in Mass Spectrometry, 8,(1990) 293-296. “Matrix-assisted Infrared-laser (2.94 μm)Desorption/Ionization Mass Spectrometry of Large Biomolecules”.

[0041] B. Spengler, et al., Rapid Communications in Mass Spectrometry,9, (1990) 301-305. “The Detection of Large Molecules in Matrix-assistedUV-laser Desorption”.

[0042] S. Berkenkamp, et al., Proceedings National Academy of SciencesUSA, 93, (1996) 7003-7007. “Ice as a matrix for IR-matrix-assisted laserdesorption/ionization: Mass spectra from a protein single crystal”.

[0043] J. Qin, et al., Analytical Chemistry, 68, (1996) 1784-1791. “APractical Ion Trap Mass Spectrometer for the Analysis of Peptides byMatrix-Assisted Laser Desorption/Ionization”.

[0044] S. Niu, et al., American Society for Mass Spectrometry, 9, (1998)1-7. “Direct Comparison of Infrared and Ultraviolet WavelengthMatrix-Assisted Laser Desorption/lonization Mass Spectrometry ofProteins”.

[0045] D. P. Little et al., Analytical Chemistry, 22, (1997), 4540-4546“MALDI on a Chip: Analysis of Arrays of Low-Femtomole to SubfemtomoleQuantities of Synthetic Oligonucleotides and DNA Diagnostic ProductsDispensed by a Piezoelectric Pipet.”

[0046] Applicants have discovered that a MALDI source may effectivelyoperate at ambient pressure and that such an apparatus is particularlyuseful for the analysis of organic molecules, such as but not limited tosmall and large organic compounds, organic polymers, organometalliccompounds and the like. Of particular interest are biomolecules andfragments thereof including but not limited to biopolymers such as DNA,RNA, lipids, peptides, protein, carbohydrates—natural and syntheticorganisms and fragments thereof such as bacteria, algae, fungi, viralparticles, plasmids, cells, and the like.

SUMMARY OF THE INVENTION

[0047] The invention is directed to a mass spectrometer having a MALDIsource which operates at atmospheric pressure (hereinafter referred toas “AP-MALDI source”). The AP-MALDI source is compatible with variousmass analyzers and solves many problems associated with conventionalMALDI sources operating under vacuum.

[0048] In one embodiment, the present invention relates to-an apparatusfor ionizing at least one analyte in a sample for delivery to a massanalysis device, comprising:

[0049] (a) an ionization enclosure including a passageway configured fordelivery of ions to the mass analysis device;

[0050] (b) means to maintain the ionization enclosure at an ambientpressure of greater than 100 mTorr;

[0051] (c) a holder configured for maintaining a matrix containing thesample in the ionization enclosure at said ambient pressure;

[0052] (d) a source of laser energy including means associated with theionization enclosure for directing the laser energy onto said matrixmaintained by the holder at the ambient pressure to desorb and ionize atleast a portion of the analyte in the sample, and

[0053] (e) means for directing at least a portion of the at least oneionized analyte into the passageway.

[0054] In another embodiment, the present invention relates to anapparatus for mass analysis of at least one analyte in a sample,comprising:

[0055] (a) an ion source having an ionization enclosure and a massanalysis device having a mass analysis enclosure, the ionizationenclosure being connected with the mass analysis enclosure through apassageway configured for delivery of ions from the ion source to themass analysis device, the ion source including:

[0056] (1) a holder configured-for maintaining a matrix containing asample in the ionization enclosure at the ambient pressure;

[0057] (2) means associated with the ionization enclosure for directinglaser energy onto a matrix maintained by the holder at the ambientpressure to desorb and ionize at least a portion the at least oneanalyte in the sample, and

[0058] (3) means for directing at least a portion of the ionized analyteinto the passageway; and

[0059] (b) means to maintain the ionization enclosure at an ambientpressure greater than 100 mTorr optionally while maintaining the massanalysis enclosure at a pressure less than 10⁻⁵ Torr.

[0060] In still another embodiment, the present invention relates to amethod for preparing for mass analysis a sample that may contain atleast one analyte, comprising:

[0061] (a) providing a matrix containing the sample; and

[0062] (b) maintaining the matrix containing the sample in a conditionof ambient pressure greater than 100 mTorr while directing laser energyonto the matrix to desorb and ionize at least a portion of the at leastone analyte, and

[0063] (c) directing at least a portion of the ionized at least oneanalyte into a mass analysis device.

[0064] In another embodiment the present invention relates to a methodfor analyzing a sample that may contain at least one analyte comprising:

[0065] (a) providing a matrix containing the sample;

[0066] (b) maintaining the sample matrix in a condition of ambientpressure greater than 100 mTorr while directing laser energy onto thematrix to desorb and ionize at least a portion of the at least oneanalyte;

[0067] (c) directing at least a portion of the ionized at least oneanalyte into a mass analysis device, and

[0068] (d) mass analyzing the portion of the at least one analyte thatis received by the mass analysis device.

[0069] In yet an another embodiment, the present invention concerns amethod for the mass spectrometric analysis of ions produced bymatrix-assisted laser desorption and ionization of at least one analytein a sample, wherein the improvement comprises conducting thematrix-assisted desorption and ionization at an ambient pressure greaterthan 100 mTorr.

[0070] In still another embodiment, the present invention concerns amass analysis apparatus including a matrix-assisted laser desorption andionization (MALDI) source and a mass analysis device that receives andanalyzes ions from the MALDI source, wherein the improvement comprisesmeans for maintaining the MALDI source at an ambient pressure greaterthan 100 mTorr during the ionization and analysis.

[0071] None of the herein above cited patents or articles teach orsuggest the present invention of an apparatus and a method to conduct aMALDI analysis at or about atmospheric pressure.

[0072] The references, articles and patents described herein are herebyincorporated by reference in their entirety. In particular the reportedMALDI references or patents, when read in conjunction with thedisclosure in the text, claims and figures of this patent application,can be adapted to obtain a large number of AP-MALDI configurations at ornear ambient pressure or at or near atmospheric pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0073]FIG. 1 shows schematic diagram of a mass spectrometer having aMALDI source which operates at ambient pressure. (See below).

[0074]FIG. 2 shows enlarged schematic diagram of a MALDI source whichoperates at ambient pressure from FIG. 1.

[0075]FIG. 3A shows total ion chromatogram of α-cyano-4-hydroxycinnamicacid matrix scanned from m/z 188 to m/z 192 obtained with a quadrupolemass spectrometer.

[0076]FIG. 3B is the mass spectrum of α-cyano-4-hydroxycinnamic acidobtained.

[0077]FIGS. 4A to 4J show selected ion monitoring (SIM) signal of m/z1061 (bradykinin) obtained with a quadrupole mass spectrometer acquiringdata every 25 microseconds. FIG. 4A is capture No. 1 at 0 seconds. FIG.4B to FIG. 4J continue at the specific capture times shown in FIGS. 4Bto 4J. The vertical axis designation on FIGS. 4A to 4J and FIGS. 5A to5J is abundance.

[0078]FIGS. 5A to 5J show selected ion monitoring (SIM) signal of m/z1900 (background) obtained with a quadrupole mass spectrometer alsoacquiring data every 25 microseconds.

[0079]FIGS. 6A and 6B show ambient pressure MALDI data of a trypticdigest of bovine cytochrome c (14 pmoles deposited on a sample stage)obtained with an ion trap mass spectrometer. FIG. 6A shows total ionchromatogram (TIC) as the laser was moved across the sample spot. FIG.6B shows a 1.25 seconds averaged scan (m/z 300-1700) acquiring dataevery 250 milliseconds.

[0080]FIG. 7 shows ambient pressure MALDI data of 100 pmoles bradykininblotted on a polyvinylidine difluoride (PVDF) membrane obtained with anion trap mass spectrometer; (upper trace) total ion chromatogram (TIC)and (lower trace) 1.25 seconds averaged scan (m/z 300-1200) acquiringdata every 250 milliseconds.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0081] Definitions

[0082] As used herein:

[0083] “Ambient pressure” refers to the existing pressure within theenclosure of the AP-MALDI apparatus. The enclosure generally may havesmall openings or ports. However, the enclosure may also be sealed. Theambient pressure is greater than 100 mTorr, and maybe much higher, suchas greater than 1 Torr, 100 Torr, 1000 Torr, 2500 Torr and at pressuresintermediate to 100 mTorr and 2500 mTorr. It is understood thatpressures above 760 Torr mean that the system is under a positivepressure.

[0084] “Atmospheric pressure” is a subset of “ambient pressure” andrefers to the normal air pressure, e.g. 760 mm Hg at sea level. Near orabout atmospheric pressure refers to pressures that are between about+15% and −15% of atmospheric pressure, preferably between about +10% and−10% more preferably between about +5% and −5%. Atmospheric pressure ismost preferred. In some cases, a positive pressure (e.g. inert gas) ison the system to control the flow.

[0085] “Ambient temperature” or “atmospheric temperature” is about 20°C. 10° C.

[0086] “Flowing” refers to a liquid sample or matrix which is moving andfrom which the sample and matrix is analyzed.

[0087] “Holder” refers to a holder for a sample and matrix in this art.Holder includes, but is not limited to, location on a surface; on or inone or more wells of a multi-well microtitre plate; on a microchiparray; on or from a thin layer chromatographic plate; on, in or from anelectrophoresis gel, on or from a membrane, or combinations thereof.“Holder” also refers to an interface for introducing a moving liquide.g., the effluent from a HPLC or CE a syringe pump and the like.

[0088] “Location of sample” refers to the situation wherein the said atleast one analyte in a matrix is located on a surface; on or in one ormore wells of a multi-well microtitre plate; microchip array; on or froma thin layer chromatographic plate; on, in or from an electrophoresisgel, on or from a membrane, or combinations thereof.

[0089] “Matrix” refers to any solid or liquid molecules having theability to transfer or receive a charge from the analyte and anabsorption at the wavelength of the laser, such as ultraviolet (UV),(electronic), visible (VIS) or infrared (IR) (vibrational and/orrotational) or combinations thereof. For an ultraviolet laser,substituted aromatic compounds are used which can transfer or receive achange to or from the analyte. For an infrared laser, aliphatic organiccompounds, hydrocarbons, aliphatic organic compounds which containheteroatoms such as oxygen, nitrogen, sulfur, and combinations thereof,water and combinations of these compounds which can transfer to orreceive a charge from the analyte are suitable.

[0090] “Means for maintaining ambient (or atmospheric) pressure” refersto methods and equipment which are currently available. These includebut are not limited to (1) a passageway and/or associated ion opticswhich restricts the gas flow from the ionization enclosure to the massanalyzer enclosure; (2) gas which is introduced to the ionizationenclosure to produce above ambient pressure and optionally aboveatmospheric pressure; (3) a gas which is introduced to the ionizationenclosure which entrains and carries the ionized analytes into thepassageway; (4) a separate pump to create the greater than 100 mTorrpressure and the like.

[0091] “Static” refers to a sample or matrix which is not moving at thetime of analysis.

[0092] In one aspect, the reference of A. Krutchinsky, et al., in RapidCommunications in Mass Spectrometry, 12, (0.1998) 508-518. “OrthogonalInjection of Matrix-assisted Laser Desorption/Ionization Ions into aTime-of-flight Spectrometer Through Collisidnal Damping Interface” is ofinterest. It discusses the effect of ion collisional damping on massanalysis at ion source pressures of 10-100 mTorr.

[0093] Construction of the AP-MALDI Source

[0094] The AP-MALDI source contains the following:

[0095] (a) a surface for depositing the matrix/analyte mixture;

[0096] (b) a laser to desorb and ionize the matrix/analyte mixture;

[0097] (c) a passageway from the AP-MALDI source to ion optics and massanalyzer/detector; and

[0098] (d) means for ions produced from the matnx/analyte mixture to beextracted are drawn into the passageway from the AP-MALDI source (suchas a potential gradient, a gas to entrain, a vacuum system to draw andthe like).

[0099] Suitable surfaces for depositing the matrix/analyte mixtureinclude a probe tip, sample stage and the like. The probe tip or samplestage may be constructed from a number of materials including metals(such as stainless steel, gold, silver, aluminum, and the like),semiconductors (e.g. silicon), and insulators (such as quartz, glass orpolymers, e.g. PDVF (or PU defined below)).

[0100] Suitable lasers include UV, VIS, and IR lasers such as nitrogenlasers, CO₂ lasers, Er-YAG lasers, Nd-YAG, Er-YTLF, Er-YSGG and thelike. Typical laser energies which are useful in AP-MALDI analysis ofbiopolymers are 10⁶-10⁸ watts/cm². Typical laser wavelengths are 200-600nm (UV-VIS wavelengths) and 1.4-12 μm (IR wavelengths), preferably 1.4-4μm.

[0101] The passageway from the AP-MALDI source to the ion optics andmass analyzer/detector may be an ion sampling orifice, capillary or thelike. The term “passageway” as used in this application, means “iontransport guide” in any form whatever. It is possible that thepassageway be of such short length relative to the opening diameter thatit may be called an orifice. Other ion transport guides includingcapillary(s), multiple ion guide(s), skimmer(s), lense(s) orcombinations thereof which are or may come to be used can operatesuccessfully in this invention.

[0102] The potential gradient may be produced by holding the probe tipor sample stage at ground potential and applying a high voltage to thepassageway; by applying a high voltage to the probe tip or sample stageand holding the passageway at ground potential; or any other arrangementwhich would establish a potential gradient between the entrance to thepassageway and the probe tip or sample stage and cause the ions producedto be drawn toward the passageway entrance.

[0103] Operation of the AP-MALDI Source

[0104] For sample preparation, the analyte may be co-crystallized withthe matrix, embedded in a layer of matrix material on a solid support,or may be deposited on top of a matrix layer. The solution containingthe dissolved analyte and matrix is applied to a probe tip or samplestage. The matrix, which may be composed of any small molecules whichabsorb energy at the wavelength of the laser, is capable of transferringcharge to the analyte following absorption. Suitable matrix materialsinclude cinnamic acid derivatives (such as α-cyano-4-hydroxycinnamicacid and sinapinic acid), dihydroxybenzoic acid derivatives (such as2,5-dihydroxybenzoic acid), nicotinic acid, sugars, glycerol, water andthe like. Suitable solvents include methanol, acetonitrile, water andthe like. The analyte matrix may be a liquid such as water or alcohole.g methanol, or a solid such as ice.

[0105] The analyte in a matrix in one embodiment is located on asurface; on or in one or more wells of a multi-well microtitre plate ora microchip array; on or from a thin layer chromatographic plate; on, inor from an electrophoresis gel, on or from an electroblotted membrane,or combinations thereof. In another embodiment, the sample holding meansis any conventional single or multi-chambered containment article. Thesampling may occur using a static or a flowing liquid sample, such asthe effluent from an HPLC, CE, or syringe pump.

[0106] The laser is operated at ultraviolet (UJV), visible (VIS), orinfrared (IR) wavelengths or combinations thereof. The operation of theAP-MALDI configuration and/or sampling occurs in air, helium, nitrogen,argon, oxygen, carbon dioxide, or combinations thereof. It is also in aninert environment selected from helium, nitrogen, argon or combinationsthereof.

[0107] As in conventional MALDI sources, a focused laser is directed andfired at the matrix/analyte mixture, thereby ionizing the analyte. Theionized cloud is drawn to the ion transport guide by the potentialgradient between the probe tip or sampling stage and the passageway. Theions enter the passageway and pass into the ion optics and massanalyzer/detector.

[0108] The operation of the AP-MALDI configuration and/or samplingoccurs in air, helium, nitrogen, argon, oxygen, carbon dioxide, orcombinations thereof, or in an inert environment selected from helium,nitrogen, argon, or combinations thereof.

[0109] Suitable mass analyzers/detectors include time-of-flight, iontrap, quadrupole; Fourier transform ion cyclotron resonance, magneticsector, electric sector, or combinations thereof.

[0110] In one application, the laser is stationary and the at least onesample are multiple samples and the multiple samples are positioned andsequentially analyzed in an organized or a random manner.

[0111] In another application, multiple samples are contained in amultiple sample holder which is stationary and the laser is mobile andis positioned to sequentially analyze the stationary multiple samples inan organized or random manner.

[0112] The AP-MALDI configuration of this invention is operable over abroad temperature range between about −196° C. to +500° C., andpreferably between about −20° and +100° C.

[0113] In one aspect, the apparatus of the claims is configured suchthat the mass analysis device is selected from the group consisting ofan ion trap operating analyzer operating at about 10⁻⁵ Torr and atime-of-flight mass spectrometer operating at about 10⁻⁶ Torr.

[0114] The method and apparatus of the invention provide a number ofadvantages over conventional MALDI and related techniques:

[0115] (1) Generating MALDI ions at ambient pressure permits easierconstruction of a rapid sample switching device. This is an importantimprovement in mass spectrometry which permits rapid, high volumeanalysis of samples using AP-MALDI as the ionization source.

[0116] (2) The laser energy employed may be greater and more variablethan for conventional MALDI-TOF systems because ions are cooled in thetransport process from atmosphere to vacuum in AP-MALDI. With AP-MALDI,ion energy spreads are much lower and the signal is more intenseresulting in higher sensitivity. As a result, the higher laser energygenerates more analyte ions and thereby improves the sensitivity of theapparatus compared to conventional systems. Furthermore, since theperformance characteristics of the laser are less critical, a lower costlaser may be employed.

[0117] (3) The relaxation of sample stage position and flatnessrequirements permits analysis of analyte directly from materials such aspolyvinylidine difluoride (hereinafter referred to as “PVDF”) membranes,polyurethane (PU) membranes, polyacrylamide gels and other materialswhich are commonly used in biological sample analysis. The ability toanalyze samples directly from or off these materials greatly reducessample handling and its associated cost.

[0118] (4) AP-MALDI may be used as an additional ionization source forother mass spectrometer systems. For example, a user could use either anAP-MALDI, API-ES (including nanospray) or APCI technique to analyzesamples on the same mass spectrometer (mass analyzer/detector) withminimal additional capital investment. Provided the multiple ionizationsource mass spectrometer had a mass range to support the predominatelysingly charged ions generated by AP-MALDI, there would be little needfor a separate MALDI-TOF instrument.

[0119] (5) Because the apparatus operates at ambient pressure, AP-MALDIis able to work with mass analyzers other than TOF, including ion trap(MS/MS) analysis. Conventional MALDI sources produce ions having a largeenergy spread, the lowest possible laser energy is used to produce ions.However, the trade-off is that the lower laser energy is inefficient inproducing ions. Since ions are cooled in the transport process fromatmosphere to vacuum in AP-MALDI, higher laser energy may be used togenerate more sample ions, as discussed above. With AP-MALDI, ion energyspreads are much lower resulting in greater ion collection efficienciesand therefore higher sensitivity.

[0120] (6) The AP-MALDI source offers advantages over nanospray ESI forbiopolymer identification. Nanospray ESI is a technique which provideshigh sensitivity and may be used to analyze limited quantities ofsamples because the samples are introduced into the mass spectrometer(mass analyzer/detector) at very low flow rates. Accordingly, theanalyst may review the spectrum of the sample and make a decision aboutany further MS or MS/MS analysis which may be necessary. The majordrawbacks of the nanospray ESI technique are that a high level of skillis needed to carry out the technique, it is difficult to stop andrestart the analysis and sample will be consumed while the analyst isdetermining what further analysis may be necessary. These drawbacks maybe reduced by using an AP-MALDI source because AP-MALDI is a pulsetechnique. As such, the analyst may generate data, analyze it and thenperform additional MS or MS/MS analysis without the loss of sample. Inaddition, AP-MALDI may be easier to operate than conventional nanospraytechniques.

Description of FIGS. 1 and 2

[0121]FIGS. 1 and 2 are a schematic representation of a cross section ofan ambient pressure MALDI source (10A) and mass spectrometer (10B).Laser (11) is activated directing a laser beam (12) to the sample in thematrix (13) on sample holder (14), at or about ambient pressure. Sampleholder (14) may be a multi-well sample plate, which is moved in anorganized manner by a conventional multi-axis (XYZ) sample translationand rotation stage (15). This stage is programmable and can operateunder data system control. Sample holder (14) is grounded (16). Samplein the matrix (13) is ionized producing ions (17) in the ambientpressure chamber (18) having cover (19). The atmosphere within thechamber (18) is usually air, however, conventional inert gases may beused to suppress oxidation of the analyte or portion thereof. All ofthese components with the exception of the laser (11) are located withinthe sample chamber mount (20). The ions produced pass through adielectric capillary (21) which is usually held at several kilovoltspotential, through a first skimmer (22), a lens (23) multiple ion guide(24) and a second skimmer (25) to be analyzed by a mass spectrometer(26). It should be understood that the above description is intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

[0122] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the apparatus and method of the invention, and arenot intended to limit the scope of what the inventors regard as theirinvention.

[0123] General

[0124] The equipment used for the present invention is conventional inthis art. For example, many vacuum pumps are commercially available froma number of suppliers such as Edwards, One Edwards Park, 301 BallardvaleStreet, Wilimington, Mass. 01887. Model EM21, double stage (2.2 m³h⁻¹,1.3 ft²m⁻¹, 37 I min⁻¹) is a small mechanical vacuum pump whichtypically operates in the 1 to 100 mTorr range or higher. Anothercommercial supplier of suitable vacuum pumps is LABOPORT. One of skillin this art can select the pumps which will achieve the vacuum orpressure levels described herein.

EXAMPLE 1 Matrix: α-cyano-4-hydroxycinnamic Acid; Analyte Bradykinin

[0125] As shown in FIG. 2, an AP-MALDI source was constructed from asample stage made from a sheet of metal and held at ground potential.The sample stage was positioned approximately 5=m opposite anatmospheric ion sampling capillary held at high voltage potential (4kV). A focused nitrogen laser of wavelength 337 nm was directed andfired at a rate of 20 Hz at a dried spot of a matrix/sample mix on thesample stage, ionizing the matrix/sample mix.

[0126] To demonstrate the formation of matrix ions, a narrow scan fromm/z 188 to m/z 192 was performed. The scan is shown in FIG. 3. Theα-cyano matrix may be detected as a [M+H]⁺ ion at m/z 190 (see FIG. 4).The presence of the m/z 191 isotope (¹³C) confirmed that ions weregenerated and that the signal was not due to a noise event.

[0127] To demonstrate the formation of analyte ions (bradykinin), thequadrupole mass filter was set to transmit ions of mass-to-charge 1061and data acquired every 25 microseconds. The data is shown in FIG. 5.Signal events substantially above background demonstrate the generationof analyte ions. To demonstrate that the signal generated at m/z 1061was actually analyte and not an artifact, data was also acquired withthe quadrupole set to transmit ions of mass-to-charge 1900. The data areshown in FIGS. 5A to 5J. The lack of a signal confirmed that the signalsin FIGS. 4A to 4J was actually from the analyte and not an artifact. InFIG. 4G the laser firings are designated as 41, 42, 43, and 44 relatedto the [M+H]+ of bradykinin.

[0128]FIGS. 6A and 6B show ambient pressure MALDI data of a trypticdigest of bovine cytochrome c (14 pmoles deposited on a sample stage).FIG. 6A shows the total ion chromatogram (tiC) as the laser was movedacross the sample spot. FIG. 6B shows 1.25 seconds averaged scan (m/z300-1700) acquiring data every 250 milliseconds.

[0129]FIG. 7 shows ambient pressure MALDI data of 100 pmoles bradykininblotted on a PVDF membrane; (upper trace) total ion chromatogram (TIC)and (lower trace) 1.25 seconds averaged scan (m/z 300-1200) acquiringdata every 250 milliseconds.

[0130] While the invention has been described and illustrated withreference to specific embodiments, those skilled in the art willrecognize that modification and variations may be made in the analysisof analytes in a sample in a matrix using a MALDI configuration atambient pressure without departing from the principles of the inventionas described herein above and set forth in the following claims.

We claim:
 1. An apparatus for ionizing at least one analyte in a samplefor delivery to a mass analysis device, comprising: (a) an ionizationenclosure including a passageway configured for delivery of ions to themass analysis device; (b) means to maintain said ionization enclosure atan ambient pressure of greater than 100 mTorr; (c) a holder configuredfor maintaining a matrix containing said sample in said ionizationenclosure at said ambient pressure; (d) a source of laser energyincluding means associated with said ionization enclosure for directingthe laser energy onto said matrix maintained by said holder at saidambient pressure to desorb and ionize at least a portion of said analytein the sample, and (e) means for directing at least a portion of said atleast one ionized analyte into said passageway.
 2. The apparatus ofclaim 1 wherein said at least one analyte in a matrix is located on asurface; on or in one or more wells of a multi-well microtitre plate; amicrochip array; on or from a thin layer chromatographic plate; on, inor from an electrophoresis gel, on or from a membrane, or combinationsthereof.
 3. The apparatus of claim 1 wherein the sample holding means isany conventional single or multi-chambered containment article.
 4. Theapparatus of claim 1 wherein the sampling occurs using a static or aflowing liquid sample.
 5. The apparatus of claim 1 wherein the massanalysis device is a mass spectrometer;
 6. The apparatus of claim 1wherein the souxce of laser energy is selected from a laser operated atultraviolet (UV), visible (VIS) or infrared (1R) wavelengths orcombinations thereof.
 7. The apparatus of claim 1 wherein the ambientpressure is atmospheric pressure.
 8. An apparatus for mass analysis ofat least one analyte in a sample, comprising: (a) an ion source havingan ionization enclosure and a mass analysis device having a massanalysis enclosure, said ionization enclosure being connected with saidmass analysis enclosure through a passageway configured for delivery ofions from the ion source to the mass analysis device, said ion sourceincluding: (1) a holder configured for maintaining a matrix containing asample in the ionization enclosure at ambient pressure; (2) meansassociated with said ionization enclosure for directing energy from alaser onto a matrix maintained by said holder at ambient pressure todesorb and ionize at least a portion of said at least one analyte in thesample, and (3) means for directing at least a portion of said ionizedanalyte into said passageway; and (b) means to maintain said ionizationenclosure at an ambient pressure greater than 100 mTorr whilemaintaining said mass analysis enclosure at a pressure less than about10⁻⁵ Torr.
 9. The apparatus of claim 8 wherein the mass .spectrometricanalyzer is selected from time-of-flight, ion trap, quadrupole, Fouriertransform ion cyclotron resonance, magnetic sector, electric sector, orcombinations thereof.
 10. The apparatus of claim 8 wherein the laser isoperated at ultraviolet (UV), visible (VIS), or infrared (IR)wavelengths or combinations thereof.
 11. The apparatus of claim 8wherein said at least one analyte in a matrix is located on a surface;on or in one or more wells of a multi-well microtitre plate; a microchiparray; on or from a thin layer chromatographic plate; on, in or from anelectrophoresis gel, on or from a membrane, or combinations thereof;wherein the sample holding means is any conventional single ormulti-chambered containment article; or the sampling occurs using astatic or a flowing liquid sample.
 12. The apparatus of claim 8 whereinin step (a), the operation of the MALDI configuration and samplingoccurs in air, helium, nitrogen, argon, oxygen, carbon dioxide, orcombinations thereof.
 13. The apparatus of claim 8 wherein the source oflaser energy is selected from a laser operated at ultraviolet (UV),visible (VIS) or (1R) infrared wavelengths or combinations thereof, andthe laser is stationary.
 14. The apparatus of claim 8 wherein theambient pressure is atmospheric pressure.
 15. A method for preparing formass analysis a sample that may contain at least one analyte,comprising: (a) providing a matrix containing said sample; and (b)maintaining the said matrix containing said sample in a condition ofambient pressure greater than 100 mTorr while directing laser energyonto the matrix to desorb and ionize at least a portion of the at leastone analyte, and (c) directing at least a portion of the ionized atleast one analyte into a mass analysis device.
 16. The method of claim15 wherein the MALDI is operated at or near ambient pressure and thesample is maintained in a cooled or heated state from between about −196to 500° C.
 17. The method of claim 15 wherein at least one analyte is anorganic compound selected from small molecules having a molecular weightof less than about 1000 daltons and synthetic organic polymers having amolecular weight of up to 1,000,000 daltons, or fragments of thesecompounds or polymers.
 18. The method of claim 15 wherein at least oneanalyte is a biologically related or biologically derived materialselected from the group consisting of deoxyribonucleic acid (DNA),ribonucleic acid (RNA), peptide, protein, lipid, carbohydrate, anorganism, a plasmid, bacteria, fungi, algae, viral particles, cells andcombinations and fragments thereof.
 19. The apparatus of claim 15wherein the source of laser energy is selected from a laser operated atultraviolet (UV), visible (VIS) or (IR) infrared wavelengths orcombinations thereof.
 20. The apparatus of claim 15 wherein the ambientpressure is atmospheric pressure.
 21. A method for analyzing a samplethat may contain at least one analyte comprising: (a) providing a matrixcontaining the sample; (b) maintaining said sample matrix in a conditionof ambient pressure greater than 100 mTorr while directing laser energyonto the matrix to desorb and ionize at least a portion of the at leastone analyte; (c) directing at least a portion of the ionized at leastone analyte into a mass analysis device, and (d) mass analyzing theportion of the at least one analyte that is received by the massanalysis device.
 22. The method of claim 21 wherein the mass analysisdevice means is selected from the group consisting of time-of-flight,ion trap, quadrupole, Fourier transform ion cyclotron resonance,magnetic sector, electric sector, and combinations thereof.
 23. Themethod of claim 21 wherein the laser is stationary and said at least onesample are multiple samples and the multiple samples are positioned andsequentially analyzed in an organized or a random manner.
 24. The methodof claim 21 wherein said at least one sample are multiple samples andare contained in a multiple sample holder which is stationary and saidlaser is mobile and is positioned to sequentially analyze the stationarymultiple samples in an organized or random manner:
 25. The method ofclaim 21 wherein the laser is operated at ultraviolet (UV), visible(VIS), or infrared (IR) wavelengths or combinations thereof.
 26. Themethod of claim 21 wherein the operation of the apparatus samplingoccurs in air, helium, nitrogen, argon, oxygen, carbon dioxide orcombinations thereof.
 27. The method of claim 21 wherein the samplingoccurs in an inert environment selected from helium, nitrogen, argon orcombinations thereof.
 28. The method of claim 21 wherein the apparatusis operated at or near ambient pressure and the at least one sample ismaintained at between about −196 to 500° C.
 29. The method of claim 21wherein positive ions and negative ions are produced and analyzed. 30.The apparatus of claim 1 wherein said apparatus is maintained at atemperature between about −20° C. and +100° C.
 31. The apparatus ofclaim 1 wherein the pressure of the apparatus is maintained betweenabout +15% and −15% of atmospheric pressure.
 32. A method for the massspectrometric analysis of ions produced by matrix-assisted laserdesorption and ionization of at least one analyte in a sample, whereinthe improvement comprises conducting the matrix-assisted desorption andionization at an ambient pressure greater than 100 mTorr.
 33. Massanalysis apparatus including a matrix-assisted laser desorption andionization (MALDI) source and a mass analysis device that receives andanalyzes ions from the MALDI source, wherein the improvement comprisesmeans for maintaining the MALDI source at an ambient pressure greaterthan 100 mTorr during the ionization and analysis.